1. Technical Field
The present invention relates to a formulation for the delivery of a variety of beneficial and/or therapeutic compounds by encapsulation within liposomes, and a machine of unique design for the controlled production of same. Specifically the invention relates to a precisely controlled metering system for the mixing of the two or more components of the liposomal preparations so that the various factors affecting the consistency, reproducibility and efficacy of the product may be monitored and controlled. The present invention also relates to a method and apparatus for the production of liposomal suspensions, emulsions, ointments and creams.
2. Background
Liposomes are lipid vesicles made of membrane-like lipid bilayers separated by aqueous layers. Liposomes have been widely used to encapsulate biologically active agents for use as drug carriers since water- or lipid-soluble substances may be entrapped within the aqueous layers or within the bilayers themselves. There are numerous variables that can be adjusted to optimize this drug delivery system. These include, the number of lipid layers, size, surface charge, lipid composition and the methods of preparation.
Liposomes have been utilized in numerous pharmaceutical applications, including injectable, inhalation, oral and topical formulations, and provide advantages such as controlled or sustained release, enhanced drug delivery, and reduced systemic side effects as a result of delivery localization.
Materials and procedures for forming liposomes are well-known to those skilled in the art and will only be briefly described herein. Upon dispersion in an appropriate medium, a wide variety of phospholipids swell, hydrate and form multilamellar concentric bilayer vesicles with layers of aqueous media separating the lipid bilayers. These systems are referred to as multilamellar liposomes or multilamellar lipid vesicles (xe2x80x9cMLVsxe2x80x9d) and have diameters within the range of 10 nm to 100 xcexcm. These MLVs were first described by Bangham, et al., J. Mol. Biol. 13:238-252 (1965). In general, lipids or lipophilic substances are dissolved in an organic solvent. When the solvent is removed, such as under vacuum by rotary evaporation, the lipid residue forms a film on the wall of the container. An aqueous solution that typically contains electrolytes or hydrophilic biologically active materials is then added to the film. Large MLVs are produced upon agitation. When smaller MLVs are desired, the larger vesicles are subjected to sonication, sequential filtration through filters with decreasing pore size or reduced by other forms of mechanical shearing. There are also techniques by which MLVs can be reduced both in size and in number of lamellae, for example, by pressurized extrusion (Barenholz, et al., FEBS Lett. 99:210-214 (1979)).
Liposomes can also take the form of unilamellar vesicles, which are prepared by more extensive sonication of MLVs, and consist of a single spherical lipid bilayer surrounding an aqueous solution. Unilamellar vesicles (xe2x80x9cULVsxe2x80x9d) can be small, having diameters within the range of 20 to 200 nm, while larger ULVs can have diameters within the range of 200 nm to 2 xcexcm. There are several well-known techniques for making unilamellar vesicles. In Papahadjopoulos, et al., Biochim et Biophys Acta 135:624-238 (1968), sonication of an aqueous dispersion of phospholipids produces small ULVs having a lipid bilayer surrounding an aqueous solution. Schneider, U.S. Pat. No. 4,089,801 describes the formation of liposome precursors by ultrasonication, followed by the addition of an aqueous medium containing amphiphilic compounds and centrifugation to form a biomolecular lipid layer system.
Small ULVs can also be prepared by the ethanol injection technique described by Batzri, et al., Biochim et Biophys Acta 298:1015-1019 (1973) and the ether injection technique of Deamer, et al., Biochim et Biophys Acta 443:629-634 (1976). These methods involve the rapid injection of an organic solution of lipids into a buffer solution, which results in the rapid formation of unilamellar liposomes. Another technique for making ULVs is taught by Weder, et al. in xe2x80x9cLiposome Technologyxe2x80x9d, ed. G. Gregoriadis, CRC Press Inc., Boca Raton, Fla., Vol. I, Chapter 7, pg. 79-107 (1984). This detergent removal method involves solubilizing the lipids and additives with detergents by agitation or sonication to produce the desired vesicles.
Papahadjopoulos, et al., U.S. Pat. No. 4,235,871, describes the preparation of large ULVs by a reverse phase evaporation technique that involves the formation of a water-in-oil emulsion of lipids in an organic solvent and the drug to be encapsulated in an aqueous buffer solution. The organic solvent is removed under pressure to yield a mixture which, upon agitation or dispersion in an aqueous media, is converted to large ULVs. Suzuki et al., U.S. Pat. No. 4,016,100, describes another method of encapsulating agents in unilamellar vesicles by freezing/thawing an aqueous phospholipid dispersion of the agent and lipids.
In addition to the MLVs and ULVs, liposomes can also be multivesicular. Described in Kim, et al., Biochim et Biophys Acta 728:339-348 (1983), these multivesicular liposomes are spherical and contain internal granular structures. The outer membrane is a lipid bilayer and the internal region contains small compartments separated by bilayer septum. Still yet another type of liposomes are oligolamellar vesicles (xe2x80x9cOLVsxe2x80x9d), which have a large center compartment surrounded by several peripheral lipid layers. These vesicles, having a diameter of 2-15 xcexcm, are described in Callo, et al., Cryobiology 22(3):251-267 (1985).
Mezei, et al., U.S. Pat. Nos. 4,485,054 and 4,761,288 also describe methods of preparing lipid vesicles. More recently, Hsu, U.S. Pat. No. 5,653,996 describes a method of preparing liposomes utilizing aerosolization and Yiournas, et al., U.S. Pat. No. 5,013,497 describes a method for preparing liposomes utilizing a high velocity-shear mixing chamber. Methods are also described that use specific starting materials to produce ULVs (Wallach, et al., U.S. Pat. No. 4,853,228) or OLVs (Wallach, U.S. Pat. Nos. 5,474,848 and 5,628,936).
A comprehensive review of all the aforementioned lipid vesicles and methods for their preparation are described in xe2x80x9cLiposome Technologyxe2x80x9d, ed. G. Gregoriadis, CRC Press Inc., Boca Raton, Fla., Vol. I, II and III (1984). This and the aforementioned references describing various lipid vesicles suitable for use in the invention are incorporated herein by reference.
Current methods of manufacturing liposomes are typically batch processes. Attempts at large scale or continuous manufacturing have largely been unsuccessful, primarily due to the problems associated with mixing an aqueous liquid phase with the lipid phase and the need to maintain the lipid phase at a relatively constant temperature.
Accordingly, there is a need for an improved method for the production of liposomes, preferably one that can produce liposomes in a continuous fashion rather than by batch methods, without the variations and uncontrolled differences which make large scale production of liposomal preparations problematic. In addition, there is a need for an improved method and apparatus for producing other liquid compositions, including but not limited to emulsions, ointments and creams. Those needs are met by the instant invention.
The present invention relates to a method for the continuous production of a composition of matter, such as lipid vesicles, by in-line mixing, said method comprising: (a) preparing a first phase, such as a lipid phase, and storing the lipid phase in a first storage means that is maintained at a set temperature; (b) preparing a second phase, such as an aqueous phase, and storing the aqueous phase in a second storage means that is maintained at a set temperature; (c) combining the lipid and aqueous phases by means of a mixing device having first and second metering systems, a pre-mixing system and a mixer, such as a static mixer, by: transferring the lipid phase from the first storage means to the first metering system by a first pressurized transfer means and transferring the aqueous phase from the second storage means to the second metering system by a second pressurized transfer means; transferring the lipid phase from the first metering system to a first inlet orifice in the pre-mixing system by a third pressurized transfer means and transferring the aqueous phase from the second metering system to a second inlet orifice in the pre-mixing system by a fourth pressurized transfer means; wherein the lipid phase and aqueous phases are transferred to the pre-mixing system with a high velocity creating turbulent flow; combining the lipid and aqueous phases in the pre-mixing system by shear mixing under conditions to insure that the lipid phase becomes fully hydrated by the aqueous phase to form a pre-mixed formulation; and transferring the pre-mixed formulation from an outlet orifice of the pre-mixing system to the mixer, such as by a fifth pressurized transfer means or other suitable connection or fitting; (d) forming a mixed formulation comprising lipid vesicles, in the mixer by causing the pre-mixed formulation to traverse the mixer; (e) optionally measuring the optical properties of the lipid vesicles; and (f) dispensing the mixed formulation from the mixer into a storage chamber, into a means for further modification of the properties of the lipid vesicles, or into a means of packaging the mixed formulation.
In a second aspect, the invention relates to lipid vesicles and other compositions of matter produced by the method of the invention.
In yet another aspect, the invention pertains to a method of producing compositions such as lipid vesicles using an in-line mixing system, where an active agent is encapsulated in either the aqueous core of the lipid vesicles, within the lipid bilayer of the lipid vesicles, or both. In still another aspect, the invention relates to lipid vesicle encapsulated active agents produced by the method of the invention.
Another aspect of the invention pertains to an apparatus for the continuous production of a composition of matter such as lipid vesicles by in-line mixing, said apparatus comprising: (a) a first phase, such as a lipid phase, storage means capable of being maintained at a set temperature and a first pressurized transfer means for transferring the lipid phase from the storage means; (b) a second phase, such as an aqueous phase, storage means capable of being maintained at a set temperature and a second pressurized transfer means for transferring the aqueous phase from the storage means; (c) a mixing device comprising: a first metering system for receiving the lipid phase from the first pressurized transfer means; a second metering system for receiving the aqueous phase from the second pressurized transfer means; a pre-mixing system for preparing a pre-mixed formulation, having a pre-mixing chamber; a third pressurized transfer means for transferring the lipid phase from the first metering system to a first inlet orifice in the pre-mixing system and a fourth pressurized transfer means for transferring the aqueous phase from the second metering system to a second inlet orifice in the pre-mixing system; a mixer, such as a static mixer, for preparing a mixed formulation comprising lipid vesicles, having a mixing chamber and an optional means for determining the optical properties of the mixed formulation; a fifth pressurized transfer means or other suitable connection or fitting for transferring the pre-mixed formulation from the outlet orifice of the pre-mixing system to the mixing chamber; and an optional means for applying ultrasonic energy to the pre-mixing chamber, the mixing chamber or both of said chambers; and (d) a dispensing means for transferring the mixed formulation from the mixing chamber into a storage chamber, or into a means for further modification of the properties of the lipid vesicles or into a means of packaging the mixed formulation.
In another aspect, the invention relates to liposomes produced by the apparatus of the invention.
Still another aspect of the invention relates to the use of the method and apparatus described herein for the manufacture of emulsions, and the emulsions produced thereby.